Compositions exhibiting ADP-ribosyltransferase activity and methods for the preparation and use thereof

ABSTRACT

Compositions characterized by ADP-ribosyltransferase activity are useful in promoting prophylactic and/or therapeutic responses as are promoted by, e.g., pertussis toxin but directed against another target antigen (e.g., a cancer-related antigen) in a mammalian patient.

BACKGROUND OF THE INVENTION

[0001] The present invention relates generally to the fields of medicineand biology. In particular, the present invention is directed towardscompositions exhibiting ADP-ribosyltransferase activity which haveprophylactic and/or therapeutic activity (e.g., in preventing cancermetastasis, preventing recurrence or reducing the incidence of cancers),as well as methods for the preparation and use thereof.

[0002] The use of surgery and radio- or chemotherapy to treat cancerinvolves the risk of serious side-effects and even death, yet frequentlyfails to produce substantive benefit. It is not surprising that thesemethods are rarely used to prevent cancer. It is clear there is a needfor better methods to prevent or cure cancer and/or ameliorate thesymptoms thereof in a patient.

[0003] Immune responses can effectively kill cells that display antigensthat mark cells as harboring a pathogen. Vaccines containing suchantigens can stimulate these desired responses and protect againstdisease with little risk. Coupling this experience with the hypothesisthat malignant cells may also present a similar marker has led manyinvestigators to search for vaccines that could prevent or cure varioustypes of cancer [McCall, C. A., Wiemer, L., Baldwin, S., & Pearson, F.C. (1989) Bio/technology 7, 231-240; Rosenburg, S. A. (1992) J. Clin.Oncol. 10, 180-199; Prehn, R. T. (1993) Proc. Natl. Acad. Sci. U.S.A.90, 4332-4333]. The successful development of such a vaccine wouldinvolve identifying preparations containing tumor-associated antigens,and learning how to prompt the immune system to properly andspecifically kill cells displaying those antigens.

[0004] Some vaccines have been spectacularly successful at preventinginfectious disease (e.g., smallpox); attempts to make other vaccineshave, to date, failed (e.g., AIDS). At times, the lack of success mayarise from a failure to elicit a proper response to an antigen, not theunavailability of a suitable antigen. These failures suggest thatmethods that control immune responses to antigens could greatly benefitthe performance of vaccines designed to prevent, treat and/or cureinfectious disease.

[0005] Similar issues face the development of cancer vaccines. Forexample, injecting irradiated tumor cells frequently fails to elicit aneffective anti-tumor response. However, injecting irradiated tumor cellspreviously transfected with genes causing production of lymphokines(e.g., GM-CSF) [Dranoff, G., Jaffee, E., Lazenby, A., Golumbeck, P.,Levitsky, H., Brose, K., Jackson, V., Hamada, H., Pardoll, D., &Mulligan, R. C. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 3539-3543] canpromote anti-tumor responses. Similar results have been obtained withtumor cells transfected to produce foreign major histocompatibilitycomplexes [Plautz, G. E., Yang, Z. Y., Wu, B. Y., Gao, X., Huang, L., &Nabel, G. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4645-4649], oradhesins, such as B7, normally found on the surface ofantigen-presenting cells [Chen, L., Ashe, S., Brady, W. A., Hellstroem,I., Hellstroem, K. E., Ledbetter, J. A., McGowan, P., & Linsley, P. S.(1992) Cell 71, 1093-1102; Schwarz, R. H. (1992) Cell 71, 1068-1068;Baskar, S., Ostrand-Rosenburg, S., Nabavi, N., Nadler, L. M., Freeman,G. J., & Glimcher, L. H. (1993) Proc. Natl. Acad. Sci. U.S.A. 90,5687-5690; Townsend, S. E., & Allison, J. P. (1993) Science. 259,368-370]. Yet another approach has been to stimulate the immune systemwith bacteria or factors derived therefrom [McCall et al., 1989, supra].

[0006] Pertussis toxin is a protein released from the bacteriumBordetella pertussis. The administration of pertussis toxin along with aproper antigen markedly enhances antigen-specific autoimmune disease[Munoz, J. J. (1988) in Pathogenesis and Immunity in Pertussis (Wardlaw,A. C., & Parton, R., Eds.) Chapter 8, pp. 173-192, John Wiley & SonsLtd., New York; Kamradt, T., Soloway, P. D., Perkins, D. L., & Gefter,M. L. (1991) J. Immunol. 147, 3296-3302] and antigen-specificdelayed-type hypersensitivity reactions, but not antigen-independentinflammatory responses [Sewell, W. A., Munoz, J. J., & Vadas, M. A.(1983) J. Exp. Med. 157, 2087-2096; Sewell, W. A., Munoz, J. J.,Scollay, R., & Vadas, M. A. (1984) J. Immunol. 133, 1716-1722]. Thereare reports [Likhite, V. V. (1983) U.S. Pat. No. 4,372,945; Minagawa,H., Kakamu, Y., Yoshida, H., Tomita, F., Oshima, H., & Mizuno, D. I.(1988) Jpn. J. Cancer Res. 79, 384-389; Minagawa, H., Kobayashi, H.,Yoshida, H., Teranishi, M., Morikawa, A., Abe, S., Oshima, H., & Mizuno,D. I. (1990) Br. J. Cancer 62, 372-375] that crude preparations of B.pertussis can cause anti-tumor responses; the factor in thesepreparations causing this effect was not identified. Others have shownthat lipopolysaccharides from B. pertussis can stimulate anti-tumorresponses [Ohnishi, M., Kimura, S., Yamazaki, M., Abe, S., & Yamaguchi,H. (1994) Microbiol. Immunol. 38, 733-739; Ohnishi.M, Kimura, S.,Yamazaki, M., Oshima, H., Mizuno, D.-I., Abe, S., & Yamaguchi, H. (1994)Br. J. Cancer 69, 1038-1042].

[0007] It is an object of the present invention to provide compositionsand methods which do not suffer from the drawbacks attendant to theheretofore-available compositions and methods. In particular, it is anobject of the present invention to provide compositions which increasethe efficacy of other compositions and methods.

SUMMARY OF THE INVENTION

[0008] In accordance with the present invention, there are providedcompositions characterized by ADP-ribosyltransferase activity. Thesecompositions are useful in promoting prophylactic and/or therapeuticresponses as are promoted by, e.g., pertussis toxin but directed againstanother target antigen (e.g., a cancer-related antigen) in a mammalianpatient.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The invention may be better understood with reference to theaccompanying drawings, in which:

[0010]FIG. 1 illustrates the protection against B16 melanoma afforded byvaccination of C57/BL mice with irradiated B16 tumor cells with orwithout coadministration of pertussis toxin (PT lots 48A and 55A);

[0011]FIG. 2 illustrates the protection against B16 melanoma afforded byvaccination of C57/BL mice with irradiated B16 melanoma cells firstincubated either with or without interferon-gamma (IFNg) and injectedeither with or without coadministration of pertussis toxin (PT);

[0012]FIG. 3 illustrates the protection against line 1 carcinomaafforded by vaccination of BALB/C mice with irradiated line 1 cells withor without coadministration of PT:

[0013]FIG. 4 illustrates the protection against line 1 carcinomatransfected to produce ovalbumin (L1-Ova) afforded by vaccination ofBALB/C mice with irradiated L1-Ova cells with or withoutcoadministration of PT;

[0014]FIG. 5 illustrates the ear-swelling response to irradiated L1-Ovacells of BALB/C mice previously vaccinated with irradiated carcinomacells with or without coadministration of PT, or spleen cells incubatedeither with or without pertussis toxin and then mixed with theanti-pertussis toxin monoclonal antibody 3CX4;

[0015]FIG. 6 illustrates the protection against L1-Ova cells afforded bya vaccination of BALB/C mice with irradiated L1-Ova cells with orwithout the conadministration of pertussis toxin, or spleen cellsincubated with or without pertussis toxin and mixed with 3CX4; and

[0016]FIG. 7 illustrates the protection against B16 melanoma afforded byvaccination of C57/BL mice with irradiated B16 cells with or without thecoadministration of pertussis toxin, or spleen cells incubated eitherwith or without pertussis toxin and then mixed with 3CX4.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0017] Pursuant to the present invention, responses as are promoted bypertussis toxin are directed against one or more target antigens byadministration to a mammalian patient of an effective amount of acomposition in accordance with the present invention characterized byADP-ribosyltransferase activity. Pertussis toxin is shown herein toincrease the efficacy of cancer vaccines. Compositions in accordancewith the present invention characterized by ADP-ribosyltransferaseactivity also potentiate the activity of other vaccines and other typesof therapeutic agents.

[0018] Pertussis toxin is a multi-subunit protein comprised of an Aprotomer consisting of a single catalytic S1 subunit, and a B oligomercontaining one S2, one S3, two S4, and one S5 subunits. The B oligomerbinds the toxin to specific receptors on target cells, thus deliveringthe S1 subunit to the cell membrane where, after it is activated, itcatalyzes the transfer of ADP-ribose from NAD to a specific cysteineresidue in specific acceptor proteins, typically the alpha subunit ofregulatory proteins, termed G-proteins, that bind guanine nucleotides[Ui, M. (1990) in ADP-Ribosylating Toxins and G Proteins (Moss, J., &Vaughan, M., Eds.) Chapter 4, pp. 45-77, American Society forMicrobiology, Washington, D.C.].

[0019] Although there are many other bacterial toxins known to catalyzeADP-ribosylations, to my knowledge there are no reports of otherbacterial toxins catalyzing the specific ADP-ribosylation reactionscatalyzed by pertussis toxin [Moss, J., & Vaughan, M., Eds. (1990)ADP-ribosylating Toxins and G Proteins, American Society forMicrobiology, Washington, D.C.]. Although a eukaryoticADP-ribosyltransferase activity has been described that adds ADP-riboseto cysteine residues, and perhaps to the same protein or residue as doespertussis toxin, its functional significance appears to be unknown[Williamson, K. C., & Moss, J. (1990) in ADP-Ribosylating Toxins and GProteins (Moss, J., & Vaughan, M., Eds.) pp. 493-510, American Societyfor Microbiology, Washington, D.C.]. If such an enzyme could bemanipulated to catalyze the same reaction as does pertussis toxin, thenit could become a functional analog of the toxin.

[0020] Other defined agents currently used to boost anti-tumor responsesdo not appear to contain the ADP-ribosyltransferase activity ofpertussis toxin. In experiments involving stimulation of delayed-typehypersensitivity (DTH) or auto-immune responses, pertussis toxin iscommonly used in addition to other adjuvants, such as complete Freund'sadjuvant or superantigens [Munoz, J. J., & Sewell, W. A. (1984) Infect.Immun. 44, 637-641; Sewell, W. A., de Moerloose, P. A.,McKimm-Breschkin, J. L., & Vadas, M. A. (1986) Cell. Immunol. 97,238-247; Kamradt et al., 1991, supra]; thus, the mechanism underlyingpertussis toxin action and these other adjuvants is different.

[0021] It is presently preferred to administer to mice an amount of theexemplary active agent sufficient to cause lymphocytosis, which servesas a positive control to demonstrate that the agent is active in vivo.Lymphocytosis may not be required for the beneficial effects, andamounts less than this amount may be sufficient for other responses[Munoz, J. J., Arai, H., Bergman, R. K., & Sadowski, P. L. (1981)Infect. Immun. 33, 820-826], including the anti-tumor response,particularly if the agent is directed towards specific cellular targets.In humans, another measure of pertussis toxin action may be moreappropriate, for example enhanced insulin secretion or glucoseclearance. A single intravenous injection of 0.5 or 1.0 ug pertussistoxin protein per kg body weight has been found to promote insulinsecretion in healthy, control humans with no clearly-evident toxic oradverse response. [Toyota, T., Kai, Y., Kakizaki, M., Sakai, A., Goto,Y., Yajima, M., & Ui, M. (1980) Tohoku J. Exp. Med. 130, 105-116]. Theagent may be administered by a variety of appropriate routes (e.g.intravenously, intraperitoneally) as long as the agent reaches cellswhich, upon intoxication, provide an anti-tumor response.

[0022] In the examples given herein, the effective dose of pertussistoxin injected intraperitoneally into mice appears to be less than 400ng per mouse (typical weight of 25 gm) As would readily be appreciatedby those skilled in the field, an optimum dose of active agent for anygiven mammalian patient may be determined empirically.

[0023] Properly administered, the effective dose for purposes of thepresent invention could be no more than or even less than the amount ofactivity contained in whole-cell pertussis vaccines. Such vaccines havebeen shown to reduce hyperglycemia in diabetics requiring high dosage ofinsulin [Dhar, H. L., Dhirwani, M. K., & Sheth, U. K. (1975) Brit. J.Clin. Pract. 29, 119-120], perhaps by increasing secretion of insulin.Although it has been claimed that an untoward event can arise from about300,000 doses of this vaccine, many doubt that these rare events arecausally related to the vaccine, or the pertussis toxin it contains.Millions of infants are still routinely immunized with such vaccines inthe United States [Cherry, J. D., Brunell, P. A., Golden, G. S., &Karzon, D. T. (1988) Pediatrics. 81(6 Part 2), 939-984]. Thus the risksfrom suitable agents should be well accepted by, e.g., cancer victimsfacing extended periods of suffering and death.

[0024] Just as intoxication of some cells by pertussis toxin can promoteanti-tumor responses, intoxication of other cells could diminish thedesired response, or cause unwanted side-effects. Thus, identifying thetarget(s) of pertussis toxin sufficient to promote desired anti-tumorresponses leads to preferred compositions and methods contemplated aswithin the scope of the present invention which more fully exploit theactivity of the compositions of the present invention. Much is knownconcerning the structure and function of pertussis toxin; this knowledgehas arisen, in large part, from efforts to better use pertussis toxin inpertussis vaccines [Sato, H., & Sato, Y. (1984) Infect. Immun. 46,415-421; Pizza, M., Covacci, A., Bartoloni, A., Perugini, M., Nencioni,L., De-Magistris, M. T., Villa, L., Nucci, D., Manetti, R., Bugnoli, M.,Giovannoni, F., Olivieri, R., Barbieri, J. T., Sato, H., & Rappuoli, R.(1989) Science. 246, 497-500; Loosmore, S. M., Zealey, G. R., Boux, H.A., Cockle, S. A., Radika, K., Fahim, R. E. F., Zobrist, G. J., Yacoob,R. K., Chong, P. C.-S., Yao, F.-L., & Klein, M. H. (1990) Infect. Immun.58, 3653-3662; Nencioni, L., Pizza, M., Bugnoli, M., De-Magistris, T.,Di-Tommaso, A., Giovannoni, F., Manetti, R., Marsili, I., Matteucci, G.,Nucci, D., Olivieri, R., Pileri, P., Presentini, R., Villa, L.,Kreeftenberg, J. G., Silvestri, S., Tagliabue, A., & Rappuoli, R. (1990)Infect. Immun. 58, 1308-1315; Burnette, W. N. (1991) in VaccineResearch: A Series of Advances, Vol. 1 (Koff, W., & Six, H. R., Eds.)Chapter 6, pp. 143-193, Marcel Dekker, Inc., New York] and to studytransmembrane signaling [Ui, 1990, supra]. Knowledge of site(s) ofaction of pertussis toxin which enhance anti-tumor effects leads tostrategies to identify therapeutic targets of pertussis toxin andimprove its efficacy.

[0025] For example, cells involved in the immune response (e.g.antigen-presenting or antigen-recognizing cells) are plausible targets.Such cells can be incubated and intoxicated with pertussis toxin exvivo, the remaining toxin neutralized with monoclonal antibodies, andthe intoxicated cells placed back in vivo.

[0026] Mutations in the B oligomer could be used to target pertussistoxin to sufficient targets. For example, the B oligomer containsmultiple binding sites with differing specificities. Two of these siteshave been identified, one in subunit S2, the other in subunit S3. Thesite in S2 appears to cause the toxin to bind to lung cilia; the site inS3 appears to cause binding to macrophages. Macrophages can contributeto immune responses. In addition, mutations in S2 and S3 can convert thebinding properties of one to the other [Saukkonen, K., Burnette, W. N.,Mar, V. L., Masure, H. R., & Tuomanen, E. I. (1992) Proc. Natl. Acad.Sci. U.S.A. 89, 118-122]. Such mutations could limit the binding ofpertussis toxin to one or the other cell type. Such mutations could beused to direct pertussis toxin away from cells which, when intoxicated,produce an undesired response, and towards cells that, when intoxicated,produce a desired response. Other alterations of pertussis toxinstructure which alter its binding properties may provide analogs withgreater efficacy and/or diminished undesired side-effects.Alternatively, antibodies or cytokines (e.g. interleukin-2) could beadsorbed, coupled covalently, or expressed as fusion-proteins withpertussis toxin or analogs containing its ADP-ribosyltransferaseactivity to deliver this activity with greater specificity than does thenaturally-occurring B oligomer.

[0027] DNA encoding for the activity of pertussis toxin could bedelivered to specific cell types. The portions of the toxin sequencerequired for its ADP-ribosyltransferase and other functions is beingrevealed by computer [Domenighini, M., Montecucco, C., Ripka, W. C., &Rappuoli, R. (1991) Molec. Microbiol. 5, 23-31], enzymatic [Krueger &Barbieri, 1994], and x-ray crystal studies of its structure [Stein, P.E., Boodhoo, A., Armstrong, G. D., Cockle, S. A., Klein, M. H., & Read,R. J. (1994a) Structure 2, 45-57; Stein, P. E., Boodhoo, A., Armstrong,G. D., Heerze, L. D., Cockle, S. A., Klein, M. H., & Read, R. J. (1994b)Struct. Biol. 1, 591-596]. The DNA sequence of the pertussis toxin genefrom Bordetella pertussis has been reported [Nicosia, A., Perugini, M.,Franzini, C., Casagli, M. C., Borri, M. G., Antoni, G., Almoni, M.,Neri, P., Ratti, G., & Rappuoli, R. (1986) Proc. Natl. Acad. Sci. U.S.A.83, 4631-4635].

[0028] Because pertussis toxin may be causing coordinated changes in theactivities of several types of cells involved in immune responses, thecompositions and methods of the present invention may profitably beemployed in combination with other approaches. For example, cyclic-AMPis thought to increase B7 expression on antigen-presenting cells[Nabavi, N., Freeman, G. J., Gault, A., Godfrey, D., Nadler, L. M., &Glimcher, L. H. (1992) Nature 360, 266-268], and expression of B7 maypromote anti-tumor responses [Chen, S.-H., Li Chen, X. H., Wang, Y.,Kosai, K.-I., Finegold, M. J., Rich, S. S., & Woo, S. L. C. (1995) Proc.Natl. Acad. Sci. U.S.A. 92, 2577-2581; Schwarz, 1992, supra; Baskar etal., 1993, supra; Townsend & Allison, 1993, supra]. Pertussis toxin canincrease cyclic-AMP, and thus may increase B7 expression. As increasingcyclic-AMP in an antigen-presenting cell likely does more than increaseexpression of B7, pertussis toxin may cause antigen-presenting cells toactivate T cells in ways that would benefit approaches based onincreasing the expression of B7.

[0029] Pertussis toxin may act at sites of antigenic stimulation byeither promoting the release of stimulatory lymphokines (e.g.,interferon-gamma) [Sewell et al., 1986, supra] or reducing the effectsof inhibitory factors. Therefore, the administration of pertussis toxinmight improve the effectiveness of smaller doses of lymphokines used topromote anti-tumor responses. If so, then, properly used, pertussistoxin might reduce dangerous side-effects associated with the use ofsuch lymphokines. Further, the toxin might improve the action of otheradjuvants that act by causing the release of lymphokines.

[0030] The mechanisms by which pertussis toxin promotes effectiveness oftumor vaccines might also enhance the efficacy of vaccines against cellsharboring pathogens such as parasites, bacteria, or viruses. Inaddition, the G-proteins modified by pertussis toxin mediate the actionsof a wide variety of extracellular effectors in many tissues [Furman, B.L., Sidey, F. M., & Smith, M. (1988) in Pathogenesis and Immunity inPertussis (Wardlaw, A. C., & Parton, R., Eds.) Chapter 7, pp. 147-172,John Wiley & Sons, New York; Bourne, H. R., Sanders, D. A., & McCormick,F. (1990) Nature 348, 125-132]. Thus, compositions exhibitingADP-ribosyltransferase activity may have therapeutic value in othersystems (e.g. diabetes) [Dhar et al., 1975, supra; Toyota et al., 1980,supra].

[0031] There is evidence that anti-IL4 antibodies enhance the promotionof DTH by pertussis toxin [Mu, H.-H., & Sewell, W. A. (1994) Immunology83, 639-645; Rosoff, P. M., Walker, R., & Winberry, L. (1987) J.Immunol. 139, 2419-2423]. Thus, antagonists of IL4 may help promote theanti-tumor effect of compositions in accordance with the presentinvention. As more is learned about the types of immune responses thatkill tumor cells, the information will suggest other potentiallybeneficial combinations of other agents and the materials and methods ofthis invention.

[0032] The antigen may be added to the composition, or it may be foundin tumor cells already in vivo. For example, tumor cells in vivo couldbe irradiated or treated with interferon-gamma with the simultaneousadministration of PT. Alternatively, methods introducing new genes intotumor cells in vivo may render them more immunogenic [Chen, S.-H. etal., 1995, supra; Sun, W. H., Burkholder, J. K., Sun, J., Culp, J.,Turner, J., Lu, X. G., Pugh, T. D., Ershler, W. B., & Yang, N.-S. (1995)Proc. Natl. Acad. Sci. U.S.A. 92, 2889-2893]. In all of these cases,pertussis toxin activity could then be used to promote an anti-tumorresponse against the in vivo cells.

[0033] In Example 1, a recombinant analog of pertussis toxin was usedwhich lacks ADP-ribosyltransferase activity, but retains a generalstructure equivalent to naturally-occurring pertussis toxin as evidencedby the ability to agglutinate erythrocytes (indicating that the Boligomer is functionally intact) and to elicit antibodies thatneutralize naturally-occurring pertussis toxin [Nencioni et al., 1990,supra]. The result in the example demonstrates that theADP-ribosyltransferase activity of pertussis toxin is required for theanti-tumor effect, but does not rule out a role for other activities ofthe toxin. For example, the B oligomer not only delivers the S1 subunitcontaining ADP-ribosyltransferase activity to cells, but also canproduce biological effects [Tamura, M., Nogimori, K., Yajima, M., Ase,K., & Ui, M. (1983) J. Biol. Chem. 258, 6756-6761; Rosoffet al., 1987,supra; Strnad, C. F., & Carchman, R. A. (1987) FEBS. Lett. 225, 16-20;Stewart, S. J., Prpic, V., Johns, J. A., Powers, F. S., Graber, S. E.,Forbes, J. T., & Exton, J. H. (1989) J. Clin. Invest. 83, 234-242].

[0034] To demonstrate that the anti-tumor effects of pertussis toxin arenot restricted to one tumor type or strain of mouse, Examples 1-3, and 8demonstrate an effect against B16 melanoma in C57BL/6 mice, and Examples4, 5, and 7 demonstrate an effect against a lung carcinoma termed line1, syngeneic to Balb/c mice [Blieden, T. M., McAdam, A. J., Foresman, M.D., Cerosaletti, K. M., Frelinger, J. G., & Lord, E. M. (1991) Int. J.Cancer Supplement, vol 6, 82-89]. The results with the lung carcinomasuggests that the effect of pertussis toxin is increased if the tumor ismade more immunogenic by the expression of a foreign protein, in thiscase chicken ovalbumin.

[0035] Examples 6-8 also demonstrate that the immune system is asufficient target of the toxin. The approach used was to take spleencells from one mouse, incubate them overnight with or without pertussistoxin, and then co-administer the spleen cells with irradiated tumorcells into a syngeneic mouse, which was then subsequently challengedwith live tumor cells. A problem that can be encountered with thisapproach is that pertussis toxin reversibly binds to the surface of thespleen cells. Thus, when cells incubated with pertussis toxin areinjected into a mouse, the toxin can be transferred from the injectedcells to cells of the recipient mouse. This process must be blocked inorder to establish that the cultured cells are in fact a sufficienttarget of the toxin. A monoclonal antibody, termed 3CX4 [Kenimer, J. G.,Kim, K. J., Probst, P. G., Manclark, C. R., Burstyn, D. G., & Cowell, J.L. (1989) Hybridoma. 8, 37-51], was therefore used to block transfer ofthe toxin to the recipient mouse.

[0036] To test the ability of the antibody to block transfer, theexperiment reported in Example 6 was performed. Spleen cells wereincubated overnight either with or without pertussis toxin, washed, andmixed with 3CX4. Irradiated tumor cells were then injected into miceeither with or without co-administration of these cells. For some mice,the toxin-treated cells were lysed by freeze-thawing prior to injection;pertussis toxin tolerates freeze-thawing [Kaslow, H. R., & Burns, D. L.(1992) FASEB J. 6, 2684-2690]. The action of the toxin and cells werethen evaluated by measuring an immune response to the tumor cells:irradiated tumor cells were injected into one ear, and the response wasdetermined by measuring the swelling of the ear. When administered withirradiated cells, the addition of toxin-treated cells increased swellingwhereas the addition of toxin-treated cells lysed by freeze thawing didnot. Similarly, in this experiment, vaccination with irradiated cellsalone was not sufficient: the addition of pertussis toxin or intacttoxin-treated cells was required. The conclusion is thus that 3CX4blocks the action of pertussis toxin bound to the membranes of spleencells.

[0037] Recently, a report [Dranoff et al., 1993, supra] demonstratedprotection of mice from B-16 melanoma using irradiated cells transfectedwith DNA causing production of GM-CSF. This report also examined theprotocols used in previous studies of protection which employed tumorcells transfected to produce other cytokines. The examination suggestedthat the lack of a critical control in these other studies created themisleading impression that these cytokines were crucial for theprotective effect. The control omitted in these previous studies wasvaccination with irradiated cells alone. Several of the examplesreported herein include this control, further confirming the utility ofthe compositions and methods of the present invention.

[0038] The invention may be better understood with reference to theaccompanying examples, which are intended for purposes of illustrationonly and should not be construed as in any sense limiting the scope ofthe present invention as defined in the claims appended hereto.

EXAMPLES

[0039] In the following examples, pertussis toxin was obtained fromeither List Laboratories or the State of Michigan Department of PublicHealth. The recombinant, inactive analog of pertussis toxin containedinactivating mutations in the S1 subunit (arg7->lys and glu129->gly)[Nencioni et al., 1990, supra]. Mice were obtained from standardcommercial sources. The hybridoma producing monoclonal antibody 3CX4[Kenimer et al., 1989, supra] was a gift from Dr. James Kenimer; theantibody was purified from ascites fluid using a protein A affinityprocedure (Pierce Biochemical Co.) The amounts of pertussis toxin andantibody are stated in terms of grams of protein determined bycolorimetric protein assay [Lowry, O. H., Rosebrough, N. J., Farr, A.L., & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275; Bensadoun, A.,& Weinstein, D. (1976) Anal. Biochem. 70, 241-250].

[0040] The B16 melanoma cell line was studied using syngeneic C57BL/6mice and was obtained from Dr. Malcolm Mitchell [Staib, L., Harel, W., &Mitchell, M. S. (1993) Can. Res. 53, 1113-1121]. The line 1 carcinomaand the subline producing ovalbumin were studied using syngeneic BALB/cmice and were a gift from Dr. John Frelinger [Blieden, et al., 1991,supra]. The cells were cultured and released from dishes as described[Blieden, et al., 1991, supra; Staib et al., 1993, supra], collected bycentrifugation, and resuspended in serum-free media prior to irradiationand/or injection.

[0041] Vaccinations were performed by administering, as separateintraperitoneal injections, either vehicle or an antigen preparationconsisting of irradiated tumor cells, and/or pertussis toxin (400 ng permouse) or carrier as shown in the examples. Either before or after thesevaccinations, the mice were challenged with live tumor cells via asubcutaneous injection in the upper back.

[0042] Examples 1-3 demonstrate that pertussis toxin enhances anti-tumorresponses against B16 melanoma.

Example 1 (MLT2)

[0043] On Day 0, 100,000 B16 cells were injected subcutaneously (SQ) inthe back. On Day 17, mice were given intraperitoneal (ip) injectionsconsisting of various combinations of phosphate-buffered saline (PBS),400 ng of pertussis toxin (PT) or recombinant, transferase-deficientpertussis toxin (rPT), and/or 300,000 irradiated B16 melanoma cells(ir-B16). On Days 26 and 38 a second and third set of ip injections weregiven. On Day 153, all surviving mice were challenged (SQ) with 100,000B16 cells. On Day 259, all surviving mice were again challenged (SQ)with 100,000 B16 cells. In this example, the B16 cells were culturedwith gamma-interferon for 24-48 hours prior to injection.

[0044] Mice were examined for physical evidence of tumor bulging outwardfrom the back or side, and the length of the tumor was recorded to thenearest cm. Typically death occurred when tumors were greater than 2 cmin length. Death was associated with clearly-evident tumor growth. Otherdata suggested that, at times, there can be incomplete tumor take incontrol animals. Thus, to better demonstrate generation of an anti-tumorresponse, survivors were subjected to the subsequent tumor challenges onDays 153 and 259.

[0045] The data for this example are summarized in Table 1 (MLT2).Pertussis toxin stimulated an anti-tumor response.

Example 2

[0046] The data for this example are shown in FIG. 1 and show pertussistoxin stimulated an anti-tumor response. Ten days prior to tumorchallenge (Day-10), six groups each containing six mice were injectedintraperitoneally (ip) with different combinations of antigen (300,000irradiated B16 cells previously treated with interferon-gamma,B16-IFNg(0.3) and 400 ng pertussis toxin (PT, from two different lotstermed 48A and 55A). On Day 0, the mice were all challenged with 100,000B16 cells not treated with gamma-interferon injected subcutaneously intothe back, just below the neck. Tumor size was scored and the date ofdeath recorded.

Example 3

[0047] The data for this example are shown in FIG. 2 and show thatpertussis toxin stimulated an anti-tumor response. Ten days prior totumor challenge (Day-10), eight groups of six mice were injected, somewith 400 ng pertussis toxin (PT), and some with different antigenpreparations: irradiated B16 cells (B16) either with or without a priortreatment with interferon-gamma (IFNg), either fresh (no notation) orfrozen (frzn), and either 300,000 (0.3) or 2,000,000 (2.0) cells. On Day0, all the mice were challenged with an SQ injection in the back of100,000 B16 cells. Tumor size was scored and the date of death recorded.A second set of mice were vaccinated as shown in the figure below, butthe vaccinations were 4 days after initiation of tumor. None of thesemice were protected from the tumor.

[0048] Examples 4 and 5 demonstrate that pertussis toxin enhancesanti-tumor responses against line 1 lung carcinoma.

Example 4

[0049] The data for this example are shown in FIG. 3 and show thatpertussis toxin stimulated an anti-tumor response against line 1 tumorcells. Fifteen days prior to challenging mice with an SQ injection of50,000 line 1 tumor cells, 12 mice were divided into two groups eachcontaining six mice. Both groups were injected (ip) with 300,000irradiated line 1 tumor cells. One group received in addition an ipinjection of 400 ng pertussis toxin. The mice were observed for tumorgrowth and the date of death recorded.

Example 5

[0050] The data for this example are shown in FIG. 4 and show thatpertussis toxin stimulated an anti-tumor response. Twelve days prior tochallenging mice with an SQ injection of 50,000 line 1 tumor cellstransfected with DNA encoding ovalbumin (L1-Ova), 18 mice were dividedinto three groups each containing six mice. One group was injected (ip)with 300,000 irradiated L1-Ova tumor cells. One group received 300,000irradiated cells and an additional ip injection of 400 ng pertussistoxin. One group received neither cells nor toxin. The mice wereobserved for tumor growth and the date of death recorded.

[0051] Examples 6-8 establish that treating spleen cells with pertussistoxin is sufficient to enhance anti-tumor effects.

Example 6

[0052] The data for this example show that the monoclonal antibodytermed 3CX4 blocks pertussis toxin action in vivo. The assay involvedmeasuring a delayed-type hypersensitivity (DTH) response to irradiatedL1-Ova tumor cells injected into the ear of a mouse. The DTH response isseen as a swelling of the ear over a period of several days. In thisexample, the swelling is expressed as the difference in thicknessbetween the ear injected with tumor cells, and the other ear which wasinjected with a solution containing ovalbumin.

[0053] Nine days prior to injection of irradiated L1-Ova cells into theear, groups of six mice were injected ip with combinations of 300,000irradiated L1-Ova cells, 400 ng pertussis toxin, or spleen cells fromother, naive BALB/c mice. The spleen cells were first incubatedovernight in RPMI tissue culture media supplemented with 10% fetalbovine serum. To some of the cultured cells was added 400 ng pertussistoxin per 10⁸ spleen cells prior to the overnight incubation. The nextday, the cells were centrifuged, the culture media removed, and freshmedia added. The anti-pertussis toxin monoclonal antibody termed 3CX4[Kenimer et al., 1989, supra] was then added to some of the cells (1 mgadded per 10⁸ cells); some of the cells were also lysed by freezethawing.

[0054] Mice were then injected, ip, with 300,000 irradiated L1-Ovacells. The mice were divided into six groups of six mice each. Thegroups received additional ip injections of either vehicle, 400 ngpertussis toxin (PT-direct), or the spleen cells incubated either withor without pertussis toxin and 3CX4. One set of mice received spleencells that were incubated with pertussis toxin followed by 3CX4 and thenwere lysed by freeze-thawing (spl. cells +PT +3CX4->FT). For clarity,the data showing the effect of freeze-thawing are extracted out of thefirst panel and shown in a second panel. The data clearly show that theeffect of PT was blocked by freeze-thawing the spleen cells. Thus, theeffect of pertussis toxin can be mediated by spleen cells altered by thetoxin in culture, it is not required to intoxicate cells of therecipient mouse.

Example 7

[0055] The data for this example are shown in FIG. 6 and show thatspleen cells incubated with pertussis toxin stimulate an anti-tumorresponse against L1-Ova tumor cells. Thirteen days prior to challengingmice with an SQ injection of 50,000 L1-Ova cells, mice were divided intogroups containing six mice, and injected ip with either antigen (300,000irradiated L1-Ova tumor cells) and/or adjuvant. The adjuvant was either400 ng pertussis toxin, or spleen cells cultured overnight with orwithout pertussis toxin, washed, and then mixed with 3CX4 as describedin Example 6. On Day 0, the mice were challenged with tumor cells; onegroup received a dose of irradiated cells in the ear as described inExample 6. The mice were evaluated for tumor growth and the date ofdeath noted.

Example 8

[0056] The data for this example are shown in FIG. 7 and show thatspleen cells incubated with pertussis toxin stimulate an anti-tumorresponse against B16 tumor cells. Fourteen days prior to challengingmice with an SQ injection of 300,000 B16 tumor cells, mice were dividedinto groups containing six mice, and injected ip with either antigen(300,000 irradiated B16 tumor cells) and/or adjuvant. The adjuvant waseither 400 ng pertussis toxin, or spleen cells cultured overnight withor without pertussis toxin, washed, and then mixed with 3CX4 asdescribed in Example 6. On Day 0, the mice were challenged with tumorcells; one group received a dose of irradiated cells in the ear asdescribed in Example 6. The mice were visually inspected for evidence oftumor growth. Those with no evidence of tumor growth were termed“tumor-free.” In this example, some tumors regressed and thenreappeared.

[0057] From the foregoing description, one skilled in the art canreadily ascertain the essential characteristics of the invention and,without departing from the spirit and scope thereof, can adapt theinvention to various usages and conditions. Changes in form andsubstitution of equivalents are contemplated as circumstances maysuggest or render expedient, and any specific terms employed herein areintended in a descriptive sense and not for purposes of limitation.

What is claimed is:
 1. A composition for use in promoting a biologicalresponse directed against a target antigen in a mammalian patient, saidcomposition comprising: an effective amount of at least one compoundcharacterized by ADP-ribosyltransferase activity as active agent; and asuitable carrier or excipient.
 2. A composition according to claim 1,wherein the active agent is selected from the group consisting ofpertussis toxin and analogs thereof exhibiting ADP-ribosyltransferaseactivity.
 3. A composition according to claim 1, further comprising acompound having an immunogenic profile characteristic of the targetantigen.
 4. A composition according to claim 3, comprising the targetantigen.
 5. A composition according to claim 1, wherein the targetantigen is a tumor-related antigen.
 6. A method for promoting abiological response directed against a target antigen in a mammalianpatient, said method comprising: administering to the patient aneffective amount of composition comprising at least one compoundcharacterized by ADP-ribosyltransferase activity as active agent and asuitable carrier or excipient.
 7. A method according to claim 6, whereinthe biological response is prophylactic.
 8. A method according to claim6, wherein the biological response is therapeutic.
 9. A method accordingto claim 6, further comprising administering to the patient a compoundhaving an immunogenic profile characteristic of the target antigen priorto, during or after administration of the composition.
 10. A methodaccording to claim 9, wherein the compound is the target antigen.